The goal of this proposal is to determine the role of the p16 family Of cyclin dependent kinase inhibitors in the genesis of human cancer. It is clear that the changes in cell growth that accompany neoplastic transformation must translate into changes in-the regulation of the cell division cycle. We have recently discovered a new motif in cell cycle control, namely regulation of cell cycle progression by inhibitors of cyclin dependent kinases (CDKs). p16 family proteins are specific inhibitors of cyclin dependent kinases (CDK4 and GDK6) that regulate passage through Gi. The p16 family currently contains two members, p16INK4A and p15INK4B. The p15 and p16 genes are tandemly arrayed at a site of frequent chromosomal abnormality in human tumors (9p21), implicating them as tumor suppressors. p15 and p16 are also frequently deleted or mutated in a broad spectrum of cancers. In this application, we propose to examine the role of p15 and p16 in the control of cell proliferation and to investigate how loss of these genes contributes to neoplastic transformation. In Specific Aim #1, we propose to isolate the complete complement of human and murine p16 family members. This will be accomplished through multiple approaches including low stringency PCR, low stringency hybridization, two-hybrid screening and protein purification/reverse genetics. In Specific Aim #2, we examine the promoter structure and transcriptional regulation of the p15 and p16 genes. In the case of p16, our purpose is to gain clues to its function in the control of normal cell proliferation. With p15, one goal is to contribute to an understanding of the TGF-beta signal transduction cascade. In Specific Aim #3, we will probe the three dimensional structure of p16 using X-ray crystallography, NMR and molecular modeling. Coupled with extensive mutagenesis, knowledge of the p16 structure should provide insight into the p16-CDK interaction. Specific Aim #4 is targeted toward investigating the role of p15 and p16 in human cancer. First, we will determine the frequency of p15 and p16 deletions and mutations in primary human tumors. Second, we will test whether p15 and/or p16 can revert the tumorigenic phenotype of transformed cells. Finally, to directly address the role of p15 and p16 in the development of cancer, we will generate knockout mice which specifically lack one of both of these genes.